DEVELOPMENT OF TETRAPOD LIMB

DEVELOPMENT OF TETRAPOD LIMB

Pattern formation is the process by which embryonic cells form ordered spatial arrangements of differentiated tissues; The ability to carry out this process is one of the most dramatic properties of developing organisms, and one that has provoked a sense of awe in scientists and laypeople alike;The ability of limb cells to sense their relative positions and to differentiate with regard to those positions has been the subject of intense debate and experimentation. How are the cells that differentiate into the embryonic bone specified so as to form an appendage with digits at one end and a shoulder at the other? (It would be quite a useless appendage if the order were reversed.) Here the cell types are the same, but the patterns they form are different.

The vertebrate limb is an extremely complex organ with an asymmetrical arrangement of parts. There are three major axes to consider, one of which is the proximal (close) to distal (far) axis. The bones of the limb, be it wing, foot, hand, or flipper, consist of a proximal stylopod (humerus/femur) adjacent to the body wall, a zeugopod (radius-ulna/tibia-fibula) in the middle region, and a distal autopod (carpal fingers/tarsals-toes)  . Originally, these structures are cartilaginous, but eventually, most of the cartilage is replaced by bone.The positions of each of the bones and muscles in the limb are precisely organized. The second axis is the anterior (front) to posterior (back) axis. Our little fingers, for instance, mark the posterior side, while our thumbs are in the anterior. In humans, it is obvious that each hand develops as a mirror image of the other. One can imagine other arrangements to exist the thumb developing on the left side of both hands but these do not occur, The third axis is the dorsal-ventral axis. The palm (ventral) is readily distinguishable from the knuckles (dorsal).In some manner, the three-dimensional pattern of the forelimb is routinely produced.The basic "morphogenetic rules" for forming a limb appear to be the same in all tetrapods(see Hinchliffe 1991). Fallon and Crosby (1977) showed that grafted pieces of reptile or mammalian limb buds can direct the formation of chick limbs, and Sessions and co-workers (1989) found that regions of frog limb buds can direct the patterning of salamander limbs, and vice versa

the regeneration of salamander limbs appears to follow the same rules as developing limbs (Muneoka and Bryant 1982). But what are these morphogenetic rules?The positional information needed to construct a limb has to function in a threedimensional coordinate system.* During the past decade, particular proteins have been identified that play a role in the formation of each of these limb axes. The proximal-distal (shoulder-finger; hip-toe) axis appears to be regulated by the fibroblast growth factor (FGF) family of proteins.The anterior-posterior (thumb-pinky) axis seems to be regulated by the Sonic hedgehog protein, and the dorsal-ventral (knuckle-palm) axis is regulated, at least in part, by Wnt7a.The interactions of these proteins determine the differentiation of the cell types and also mutually support one another.

Formation of the Limb Bud

Specification of the limb fields: Hox genes and retinoi c acid;Limbs will not form just anywhere along the body axis. Rather, there are discrete positions where limb fields are generated.researchers have precisely localized the limb fields of many vertebrate species. Interestingly, in all land vertebrates, there are only four limb buds per embryo, and they are always opposite each other with respect to the midline. Although the limbs of different vertebrates differ with respect to which somite level they arise from, their position is constant with respect to the level of Hox gene expression along the anterior-posterior axis.The lateral plate mesoderm in the limb field is also special in that it will induce myoblasts to migrate out from the somites and enter the limb bud. No other region of the lateral plate mesoderm will do that,Retinoic acid appears to be critical for the initiation of limb bud outgrowth, since blocking the synthesis of retinoic acid with certain drugs prevents limb bud initiation (Stratford et al. 1996). Bryant and Gardiner (1992)suggested that a gradient of retinoic acid along the anterior-posterior axis might activate certain homeotic genes in particular cells and there by  specify them to become included in the limb field. The source of this retinoic acid is probably Hensen's node (Hogan et al. 1992). The specification of limb fields by retinoic acid-activated Hox genes might explain a bizarre observation made by Mohanty-Hejmadi and colleagues (1992) and repeated by Maden (1993).

Induction of the early limb bud: Fibroblast growth factorsLimb development begins when mesenchyme cells proliferate from the somatic layer of the limb field lateral plate mesoderm (limb skeletal precursors) and from the somites (limb muscle precursors; Figure 16.3) These cells accumulate under the epidermal tissue to create a circular bulge called a limb bud.            Recent studies on the earliest stages of limb formation have shown that the signal for limb bud formation comes from the lateral plate mesoderm cells that will become the prospective limb mesenchyme.

These cells secrete the paracrine factor FGF10. FGF10 is

capable of initiating the limb-forming interactions between

the ectoderm and the mesoderm. If beads containing

FGF10 are placed ectopically beneath the flank ectoderm,

extra limbs emerge (Figure 16.4) (Ohuchi et al. 1997;Sekine et al. 1999).

Specification of forelimb or hindlimb: Tbx4 and Tbx5

In 1998 and 1999, however, several laboratories (Ohuchi et al. 1998; Logan et al. 1998; Takeuchi et al. 1999; Rodriguez-Esteban et al. 1999, among others) provided gain-of-function evidence that Tbx4 and Tbx5 specify hindlimbs and forelimbs, respectively.First, if FGFsecreting beads were used to induce an ectopic limb between the chick hindlimb and forelimb buds, the type of limb produced was determined by the Tbx protein expressed. Those buds induced by placing FGF beads close to the hindlimb (opposite somite 25) expressed Tbx4 and became hindlimbs. Those buds induced close to the forelimb (opposite somite 17) expressed Tbx5 and developed as forelimbs (wings).Those buds induced in the center of the flank tissue expressed Tbx5 in the anterior portion of the limb and Tbx4 in the posterior portion of the limb.These limbs developed as chimeric structures, with the anterior resembling a forelimb and the posterior resembling a hindlimb (Figure 16.5). Moreover, when a chick embryo was made to express Tbx4 throughout the flank tissue (by infecting the tissue with a virus that expressed Tbx4), limbs induced in the anterior region of the flank often became legs instead of wings;Thus, Tbx4 and Tbx5 appear to be critical in instructing the limbs to becomehindlimbs and forelimbs, respectively.

Induction of the apical ectodermal ridge

As mesenchyme cells enter the limb region, they secrete factors that induce the overlying ectoderm to form a structure called the apical ectodermal ridge (AER) (Figure 16.7; Saunders 1948; Kieny 1960; Saunders and Reuss 1974).                                           This ridge runs along the distal margin of thelimb bud and will become a major signaling center for the developing limitIts roles include;

1. maintaining the mesenchyme beneath it in a

plastic, proliferating phase that enables the linear (proximal-distal) growth of the limb;

2. maintaining the expression of those molecules that generate the anteriorposterior (thumb-pinky) axis;

 3. interacting with the proteins specifying the anteriorposterior and dorsal-ventral axes so that each cell is given instructions on how to differentiate;The factor secreted by the mesenchyme cells to induce the AER is probably FGF10;Other FGFs, such as FGF2, FGF4, and FGF8, will also

    induce an AER to form; but FGF10 appears to be the FGF synthesized at the appropriate time and in the appropriate places.

    FGF10 is capable of inducing the AER in the competent ectoderm between the dorsal and ventral sides of the embryo

Generating the Proximal-Distal Axis of the Limb

The apical ectodermal ridge: The ectodermal component

The proximal-distal growth and differentiation of the limb bud is made possible by a series of interactions between the limb bud mesenchyme and the AER (Figure 16.8; Harrison 1918; Saunders 1948). These interactions were demonstrated by the results of several experiments on chick embryos:

1. If the AER is removed at any time during limb development, further development of distal limb skeletal elements ceases

2. If an extra AER is grafted onto an existing limb bud, supernumerary structures are formed, usually toward the dib.stal end of the limb

3. If leg mesenchyme is placed directly beneath the wing AER, distal hindlimb structures (toes) develop at the end of the limb. (However, if this mesenchyme is placed farther from the AER, the hindlimb mesenchyme becomes integrated into wing structures.

4. If limb mesenchyme is replaced by nonlimb mesenchyme beneath the AER, the AER regresses and limb development ceases.

The progress zone: The mesodermal component

The proximal-distal axis is defined only after the induction of the apical ectodermal ridge by the underlying mesoderm. The limb bud elongates by means of the proliferation of the mesenchyme cells underneath the AER.  This region of cell division is called the progress zone, it extends about 200 μm in from the AER. Molecules from the AER are thought to keep the progress zone mesenchyme cells dividing, and it is now thought that FGFs are the molecules responsible When the mesenchyme cells leave the progress zone, they differentiate in a regionally specific manner. The first cells leaving the progress zone form proximal (stylopod) structures;                                                those cells that have undergone numerous divisions in the progress zone become the more distal structures

The mitotic state of the progress zone is maintained by interactions between the FGF proteins of the progress zone and of the AER. FGF10 secretion by the             mesenchyme cells induces the AER, and it also induces the AER to express FGF8 (Figure 16.12). The FGF8 secreted by the AER reciprocates by maintaining the mitotic activity of the progress zone mesenchyme cells

Hox genes and the specification of the proximal-distal axis

The type of structure formed along the proximal-distal axis is specified by the Hox genes.The products of the Hox genes have already played a role in specifying the place where the limbs will form. Now they will play a second role in specifying whether a particular mesenchymal cell will become stylopod, zeugopod, or autopod.The mechanism by which Hox genes could specify the proximal-distal axis is not yet understood, but one clue comes from the analysis of chicken Hoxa-13. Ectopic expression of this gene (which is usually expressed in the distal ends of developing chick limbs) appears to make the cells expressing it stickier. This, in turn, would cause the cartilaginous nodules to condense in specific ways

As the limb grows outward, the pattern of Hox gene expression changes. When the stylopod is forming, Hoxd-9 and Hoxd-10 are expressed in the progress zone mesenchyme. When the zeugopod bones are being formed, the pattern shifts remarkably, displaying a nested sequen The posterior region expresses all the Hoxd genes from Hoxd-9 to Hoxd-13, while only Hoxd-9 is expressed anteriorly. In the third phase of limb development, when the autopod is forming, there is a further redeployment of Hox gene products. Hoxd-9 is no longer expressed.Rather, Hoxa-13 is expressed in the anterior tip of the limb bud and in a band marking the boundary of the autopod. Hoxd-13 products join those of Hoxa-13 in the anterior region of the limb bud, while Hoxa-12,Hoxa-11, and Hoxd-10 12 are expressed throughout the posterior twothirds of the limb bud.ce of Hoxd gene expression.

Specification of the Anterior-Posterior Limb Axis;

The zone of polarizing activity

The specification of the anterior

posterior axis of the limb is the earliest

change from the pluripotent condition.

Although the differentiation of the

proximal-distal structures is thought to

depend on how many divisions a cell 

undergoes while in the progress zone, 

information instructing a cell as to its 

position on the anterior-posterior and 

dorsal-ventral axes must come from

other   sources.    Several  experiments (Saunders and Gasseling 1968; Tickle et al. 1975) suggest that the anterior-posterior  axis is specified by a small block of mesodermal tissue near the posterior junction of the young limb bud and the body wall. This region of the mesoderm has been called the zone of polarizing activity (ZPA)When this tissue is taken from a young limb bud and transplanted into a position on the anterior side of another limb bud , the number of digits of the resulting wing  isdoubled. Moreover, the structures of the extra set of digits are mirror images of the normally produced structures. The polarity has been maintained, but the information is now coming from both an anterior and a posterior direction.

The sonic hedgehog gene appears to be activated by an FGF protein coming from the newly formed apical ectodermal ridge.

FGF8 is secreted from the AER, and is capable of activating sonic hedgehog;The evidence suggests that the Hoxb-8 protein is involved in specifying the domain of sonic hedgehog expression and thus establishing the ZPA

The action of sonic hedgehog

When Sonic hedgehog was first shown to define the ZPA, it was thought to act as a morphogen. In other words, it was thought to diffuse from the ZPA where it was being synthesized and to form a concentration gradient from the posterior to the anterior of the limb bud. However, recent research has provided evidence that Sonic hedgehog protein (or its active amino terminal region) does not diffuse outside the ZPA (Yang et al. 1997). It is now thought that Sonic hedgehog works by initiating and sustaining a cascade of other proteins, such as BMP2 and BMP7 (Laufer et al. 1994; Kawakami et al. 1996; Drossopoulou et al. 2000). A gradient of BMPs may emanate from the ZPA and specify the digits.

The Generation of the Dorsal-Ventral Axis

The third axis of the limb distinguishes the dorsal limb (knuckles, nails) from the ventral limb (pads, soles). In 1974, MacCabe and co-workers demonstrated that the dorsal-ventral polarity of the limb bud is determined by the ectoderm encasing it.The experiment done suggested that the late specification of the dorsalventral axis of the limb is regulated byits ectodermal component. One molecule that appears to be particularly important in specifying the dorsal-ventral polarity is Wnt7a. The Wnt7a gene is expressed in the dorsal (but not the ventral) Wnt7a induces activation of the Lmx1 gene in the dorsal mesenchyme, and this gene encodes a transcription factor that appears to be essential for specifying dorsal cell fates in the limb. If this factor is expressed in the ventral mesenchyme cells, they develop a dorsal phenotype

Coordination among the Three Axes

The three axes of the tetrapod limb are all interrelated and coordinated. Some of the principal interactions among the mechanisms specifying the axes are shown in previous slides Indeed, the molecules that define one of these axes are often used to maintain another axis. For instance, Sonic hedgehog in the ZPA activates the expression of the Fgf4 gene in the posterior region of the AER . Fgf4 expression is important in recruiting mesenchyme cells into the progress zone, and it is also critical in maintaining the expression of Sonic hedgehog in the ZPA (Li and Muneoka 1999). Therefore, the AER and the ZPA mutually support each  other through the positive loop of Sonic hedgehog and  FGF4 Also the findings showed that the synthesis of Sonic hedgehog is stimulated by the combination of FGF4 and Wnt7a proteins

Cell Death and the Formation of Digits and Joints;                 

      Sculpting the autopodCell death plays a role in sculpting the limb. Indeed, it is essential if our joints are to form and if our fingers are to become separate (Zaleske 1985). The death (or lack of death) of specific cells in the vertebrate limb is genetically programmed and has been selected for during evolution.One such case involves the webbing or nonwebbing of feet.example chickens and ducks Between the time when the cell's death is determined and when death actually takes place, levels of DNA, RNA, and protein synthesis in the cell decrease dramatically In addition to the interdigital necrotic zone, there are three other regions that are "sculpted" by cell death. The ulna and radius are separated from each other by an interior necrotic zone, and two other regions, the anterior and posterior necrotic zones, further shape the end of the limb (Figure 16.22B; Saunders and Fallon 1966). Although these zones are said to be "necrotic," this term is a holdover from the days when no distinction was made between necrotic cell death and apoptotic cell death . These cells die by apoptosis, and the death of the interdigital tissue is associated with the fragmentation of their DNA The signal for apoptosis in the autopod is probably provided by the BMP proteins.BMP2, BMP4, and BMP7 are each expressed in the interdigital mesenchyme, and blocking BMP signaling (by infecting progress zone cells with retroviruses carrying dominant negative BMP receptors) prevents interdigital apoptosis.This suppression may come from the Noggin protein, which is made in the developing digits and in the perichondrial cells surrounding them If noggin is expressed throughout the limb bud, no apoptosis is seen.

Forming the joints

The function originally

as decribed to BMP 

was the formation, 

the another prevention

Of bone and cartilage 

tissue. In 

the developing limb,

BMPs induce

the mesenchymal cells either to undergo apoptosis or to become cartilage

producing chondrocytes depending on the stage of development. The same BMNs

can induce death or differentiation, depending on the age of the target cell. This "context dependency" of signal action is a critical concept in developmental biology. "constantia" , serif; font-size: 13.5pt;"> It is also critical for the formation of joints. Macias and colleagues (1997) have shown that during early limb bud stages (before cartilage condensation), beads secreting BMP2 or BMP7 cause apoptosis

.Two days later, the same beads cause the limb bud cells to form cartilage.In the normally developing limb, BMPs use both these properties to form joints. BMP7 is made in the perichondrial cells surrounding the condensing chondrocytes and promotes cartilage formation.Two other BMP proteins, BMP2 and GDF5, are expressed at the regions between the bones, where joints will form The roles of BMP2 and GDF5 are more controversial. They may either be destroying mesenchymal cells to form the joint or inducing them to rapidly differentiate and join one or the other cartilaginous nodule..In either way, a space is made between the nodules, and a joint can form.

 REFERENCES

Gilbert,S.F

(2013).Develepmental Biology,Sanderland Mass,Sineaur Association Inc
Slack,J.M.W(2013).Essential Develepmental Biology OXFORD, Willey-Blackwell
Wolpert.l and Tickle.c (2011),Principles of Develepmental Oxford and New York University Press